Cloned (Comment) | Organism |
---|---|
recombinant expression of wild-type and mutant enzyme domains in Escherichia coli strain BL21(DE3) | Brucella sp. |
Crystallization (Comment) | Organism |
---|---|
Brucella LOV-HK histidine kinase domain free or in complex with non-hydrolyzable ATP analogue AMP-PCP, mixing of 1 mg/ml protein in 50 mM MES, pH 6.5, and 250 mM sodium chloride, with precipitation solution, X-ray diffraction structure determination and analysis at 2.51 A resolution, molecular modeling | Brucella sp. |
Protein Variants | Comment | Organism |
---|---|---|
A287C/L333C | site-directed mutagenesis, the mutant cross-linked dimer shows altered autophosphorylation activity compared to the wild-type enzyme | Brucella sp. |
I286C | site-directed mutagenesis of the long HK domain version, the mutation is located in the alpha1 helix N-terminal to the histidine phosphoacceptor, the mutant cross-linked dimer shows altered autophosphorylation activity compared to the wild-type enzyme | Brucella sp. |
I286C/N388A | site-directed mutagenesis of the short HK domain version, the N388A mutation impairs the phosphorylation capacity, the mutant is unable to bind ATP | Brucella sp. |
additional information | generation of two different versions of the HK domain: one long (L, the HK domain plus a 23-kDa tag, 49 kDa) and other short (S, 26 kDa). The short version has the N388A mutation which impairs the phosphorylation capacity. Both versions have the I286C mutation | Brucella sp. |
V282C | site-directed mutagenesis, the mutation is located in the alpha1 helix N-terminal to the histidine phosphoacceptor, the mutant cross-linked dimer shows altered autophosphorylation activity compared to the wild-type enzyme | Brucella sp. |
General Stability | Organism |
---|---|
DTT stabilizes | Brucella sp. |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Brucella sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + protein L-histidine | Brucella sp. | - |
ADP + protein N-phospho-L-histidine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Brucella sp. | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzyme domains from Escherichia coli strain BL21(DE3) by ultracentrifugation, nickel affinity chromatography, dialysis, and gel filtration | Brucella sp. |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + protein L-histidine = ADP + protein N-phospho-L-histidine | catalytic mechanism for Brucella LOV-HK, molecular modeling and simulations, overview | Brucella sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + protein L-histidine | - |
Brucella sp. | ADP + protein N-phospho-L-histidine | - |
? | |
additional information | autophosphorylation occurs when the ATP molecule bound to the the catalytic and ATP-binding subdomain phosphorylates the histidine residue in the dimerization/histidine phosphoacceptor subdomain. Histidine kinase dimerization implies that the directionality of the autophosphorylation reaction can proceed either in a cis (intramolecular) or in a trans (intermolecular) manner. Autophosphorylation occurs in cis in the dimer. Intramolecular interactions in the enzyme, overview. Arg321 from the alpha2 helix of the dimerization/histidine phosphoacceptor subdomain contacts Glu384, Tyr383 and the gamma phosphate from non-hydrolyzable ATP analogue AMP-PCP, closing the binding pocket | Brucella sp. | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | the Brucella HK domain presents two different dimeric assemblies in the asymmetric unit: one similar to canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. The C dimer is the functionally relevant species and can be stabilized and is active in solution. The HK domain is a monomer in solution and presents autokinase activity | Brucella sp. |
More | Brucella LOV-HK comprises an N-terminal blue-light sensor (LOV) domain, followed by a central PAS domain and a C-terminal HK domain. Crystal structure analysis, overview | Brucella sp. |
Synonyms | Comment | Organism |
---|---|---|
light-activated histidine kinase | - |
Brucella sp. |
LOV-HK | - |
Brucella sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Brucella sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Brucella sp. |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Brucella sp. |
General Information | Comment | Organism |
---|---|---|
evolution | the Brucella histidine kinase domain belongs to the HWE histidine kinase family. The HWE HK domains are present in LOV-HKs and bacteriophytochromes among other proteins | Brucella sp. |
additional information | the HK domain is a monomer in solution and presents autokinase activity | Brucella sp. |
physiological function | in response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism | Brucella sp. |